(1) Field of the Invention
The present invention relates to a novel colored molecular weight marker having a broad applicable molecular weight range. More particularly, the present invention relates to a broad-range molecular weight marker enabling measurement of molecular weights in a very broad range of from very low molecules to macromolecules.
(2) Description of the Prior Art
In the fields of biochemistry and clinical assay, an SDS gel electrophoresis method and a molecular sieve chromatography method have recently attracted attention as means for separation, purification and analysis of proteins. According to the SDS gel electrophoresis method, a polyacrylamide containing SDS (sodium dodecyl sulfate or sodium lauryl sulfate) is used as a support and the molecular weight of a protein is measured according to the electrophoresis utilizing the molecular sieve action of the polyacrylamide. This method was first reported by Sapiro et al. and Weber, Osborn (J. Biol. Chem., 244, page 4406, 1969) and Neville et al. then proposed improvements of this method.
When a protein is treated with SDS, the protein is dissociated into subunits and the subunits are surrounded by SDS to form an SDS-protein composite. Accordingly, the protein comes to have a negative charge while the protein loses its inherent charge. Since SDS is coupled with the protein at a substantially constant rate, the negative charge density of the protein becomes constant.
When the electrophoresis is carried out by using a polyacrylamide gel having a molecular sieve action as a support, the protein migrates toward the anode and the mobility of the protein depends only on the size of the protein molecule. Therefore, measurement of the molecular weight of a protein is based on the principle in which the migration distance of a protein-SDS composite in an SDS-containing gel is measured, the measured distance is compared with the migration distance of a reference protein having a known molecular weight, the relative migration distance is calculated and the molecular weight is determined from the calculated relative migration distance.
According to this method, proteins can be separated and purified by utilizing the difference of the molecular weights and the molecular weight of a protein to be analyzed can be determined from the migration distance of a reference protein in the gel. Accordingly, this method is very valuable as a means for analyzing proteins.
Crosslinked dextran developed by Porath and Flodin (supplied under the tradename "Sephadex" by Pharmacia Co., Sweden) is now available very easily, and separation of proteins by using Sephadex is now one of the basic techniques in the field of biochemistry. According to this method, substances can be fractionated based on the difference of protein molecular weights, and this method is ordinarily called "molecular sieve chromatography". This method also is used for determination of the molecular weights, and the method of P. Andrews [Journal of Biochemistry, 91, page 222 (1964)] is utilized. As the support, not only Sephadex but also gel particles of polyacrylamide and agarose have been developed and are now commercially available.
Each of the particulate gels used for molecular sieve chromatography has a three-dimensional crosslinked structure which has a certain mesh size distribution. Accordingly, molecules having a size larger than the size of all the meshes are completely inhibited from intruding into particles while molecules having a size smaller than all the mesh sizes are allowed to freely diffuse into particles. The migration speed in the gel is determined according to the coefficient of distribution between the two phases, and the smaller is the size of the molecule, the lower is the migration speed in the gel. Furthermore, molecules are dissolved out in the order of the molecule size. Therefore, according to this method, various proteins can be separated and purified based on differences of molecular weights and simultaneously, the molecular weight of a protein having an unknown molecular weight can be determined by comparing the amount of this protein dissolved out with the amount dissolved out of a reference protein having a known molecular weight.
When proteins are separated and purified according to the above-mentioned SDS electrophoresis method and molecular sieve chromatography method, determination of molecular weights of various proteins is indispensable, and if a reference protein having a known molecular weight is used, the molecular weights of various intended proteins can be determined by comparing the migration distances of these proteins with that of the reference protein.
The reference protein used for this purpose is ordinarily called "molecular weight marker". Conventional molecular markers are prepared by combining various colorless proteins differing in their molecular weight, which have been separated from natural substances. If a colorless protein is used, the operation of measuring the molecular weight cannot be performed under naked eye observation. Furthermore, a complicated operation of coloration is necessary in the final stage, with the result that a long time is required for completion of the measurement of the molecular weight.